Abstract:
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for compositions and methods that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. For example, disclosed herein are methods of modifying a target endogenous nucleic acid sequence in a nucleus of a eukaryotic cell in vitro or ex vivo, comprising: preparing a composition comprising a recombinant Cas9 protein, a single guide RNA, and a Cas9/RNA complex formed by at least a part of said recombinant Cas9 protein and single guide RNA, and introducing a Cas9/RNA complex into the eukaryotic cell in vitro or ex vivo, wherein in the composition, the single guide RNA is at least two-fold molar excess over the recombinant Cas9 protein, wherein the Cas9/RNA complex is a combination of the recombinant Cas9 protein and the single guide RNA, and wherein the Cas9/RNA complex is complexed prior to being introduced into the eukaryotic cell; wherein the single guide RNA is in vitro transcribed RNA or synthetic RNA, wherein the combination of the recombinant Cas9 protein and the single guide RNA produces a modification of the target endogenous nucleic acid sequence in the nucleus of the eukaryotic cell.
Abstract:
The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.
Abstract:
The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.
Abstract:
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides compositions for inducing targeted disruption of endogenous genes in eukaryotic cells. The composition may comprise a Cas9/guide RNA complex with a Cas9 protein to which a NLS is linked, and a guide RNA having a crRNA and a tracrRNA. The crRNA may comprise i) a portion to be hybridized with a portion of the tracrRNA, and ii) a portion complementary to a target DNA of the endogenous genes. In some embodiments, the Cas/guide RNA complex may be formed in vitro before being introduced into the eukaryotic cell.
Abstract:
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for compositions and methods that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. For example, disclosed herein is a system for inducing targeted disruption of endogenous genes in a eukaryotic cell, the system comprising: an extracellular Cas9/RNA complex; cell-free and a buffer; wherein the extracellular Cas9/RNA complex comprises: a recombinant Cas9; and a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA); wherein the extracellular Cas9/RNA complex is disposed in the cell-free buffer and wherein the extracellular Cas9/RNA complex is complexed prior to being introduced into the eukaryotic cell; and wherein the Cas9/RNA complex functions as an endonuclease that induces targeted disruption of the target DNA upon introduction into the eukaryotic cell.
Abstract:
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for compositions that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. The composition may comprise a single-chain guide RNA (sgRNA) and a Streptococcus pyogenes Cas9 protein. In some embodiments, the sgRNA and the Cas9 protein may be present in a molar ratio ranging from 29:14.0 to 29:1.4.
Abstract:
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for Cas9/RNA complexes that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. The Cas9/RNA complex may comprise a recombinant Cas9 protein including a nuclear localization signal (NLS) and a guide RNA including a crRNA and a tracrRNA. The Cas9/RNA complex may be a combination of the recombinant Cas9 protein and the guide RNA. The guide RNA may be transcribed in vitro or synthesized chemically. The target endogenous nucleic acid sequence may include a portion complementary to the crRNA of the guide RNA.
Abstract:
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for Cas9/RNA complexes that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. The Cas9/RNA complex may comprise a recombinant Cas9 protein including a nuclear localization signal (NLS) and a guide RNA including a crRNA and a tracrRNA. The Cas9/RNA complex may be a combination of the recombinant Cas9 protein and the guide RNA. The guide RNA may be transcribed in vitro or synthesized chemically. The target endogenous nucleic acid sequence may include a portion complementary to the crRNA of the guide RNA.
Abstract:
The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.
Abstract:
The present invention relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present invention relates to a composition for cleaving a target DNA in eukaryotic cells or organisms comprising a guide RNA specific for the target DNA and Cas protein-encoding nucleic acid or Cas protein, and use thereof.